The present invention relates to newly identified vascular endothelial growth factor receptor polynucleotides, their encoded polypeptides, and uses and production of such polynucleotides and polypeptides.
Formation of new blood vessels occurs as a result of two processes: vasculogenesis, which is the formation of blood vessels from progenitor cells, and angiogenesis, which is the formation of new blood vessels from preexisting vessels. Vascular endothelial growth factor (VEGF) is a secreted glycoprotein that induces angiogenesis and plays a central role in the regulation of vasculogenesis. It is highly specific for vascular endothelial cells (Dvorak et al., Am. J. Pathol. (1995) 146:1029-1039). VEGF is also known as vascular permeability factor (VPF) because of its permeabilizing effect on blood vessels. In addition to its role in the development of the vascular system, VEGF has been found to be involved in the differentiation of endothelial cells (Carmeliet et al., Nature (1996) 380:435-439; Ferrara et al., Nature, (1996) 380:439-442), cell migration, and apoptosis inhibition.
Deregulated VEGF expression contributes to the development of solid tumors by promoting tumor angiogenesis (Folkman, Nature Med. (1995) 1:27-31) and to the etiology of several additional diseases that are characterized by abnormal angiogenesis, such as metastasis, diabetic retinopathy, rheumatoid arthritis (Ferrera, Breast Cancer Res. Treat. (1995) 127-137), and abnormal wound healing (Brown et al., J. Exp. Med. (1992) 176:1375-1379).
Antibodies against VEGF can suppress tumor growth in vivo (Kim et al., Nature (1993) 362:841-844), indicating that VEGF antagonists could have broad therapeutic applications. VEGF molecules bind tyrosine kinase receptors known as VEGF receptors (VEGFRs). Three high affinity VEGFRs have been characterized in vertebrates, all of which are mainly expressed in vascular endothelial cells: VEGFR-1/FLT-1 (Yoshida et al., Cytogenet. Cell Genet. (1987) 46:724; Fong et al., Nature (1995) 376:65-69), VEGFR-2/KDR/FLK-1 (Terman et al., Oncogene (1991) 6:1677-1683; Matthews etal., Proc. Nat. Acad. Sci., (1991) 88:9026-9030), and VEGFR-3/FLT4 (Alitalo et al., U.S. Pat. No. 5,776,755; Joukov et al., EMBO J., (1996) 15:290-298). VEGFR-1, VEGFR-2, and VEGFR-3 are members of the PDGF (platelet derived growth factor) receptor family (Yarden and Ulirich, Ann. Rev. Biochem. (1988) 57: 443-478). An interesting aspect of current VEGFR biology is the perceived importance of a soluble form of VEGFR (sVEGFR), the extracellular domain without the transmembrane or intracellular domain, as an antagonist of VEGF action.
Members of signaling pathways are used reiteratively throughout evolution. For example, members of the Fibroblast Growth Factor (FGF) pathway are used in the same manner and for the same purpose, namely patterning branching morphogenesis of the respiratory system, by both insects and mammals (Metzger R J, and Krasnow M A, Science (1999) 284:1635-1639). There is a growing body of information regarding the modular subunits and the high-resolution structure of VEGF family members. Several different VEGF genes and their receptors have been identified in vertebrates. Genes from Caenorhabditis elegans encoding tyrosine kinase receptors sharing structural features with mammalian VEGFRs have been reported (Popovici et al., 1999 International Worm meeting abstract 680).
There is a clear need for a better understanding of the genetic pathways that VEGF gene family members are involved in. Further knowledge of the genetic pathways that involve or interact with VEGF as well as interacting pathway members and their collective functions and dysfunctions, could be used to develop therapeutics specifically targeted to the disease. The use of invertebrate model organism genetics and related technologies can greatly facilitate the elucidation of biological pathways (Scangos, Nat. Biotechnol. (1997) 15:1220-1221; Margolis and Duyk, Nature Biotech. (1998) 16:311). Invertebrate model organisms can also be used in the screening of putative pharmaceutical agents that are specifically targeted to a gene of interest. The identification of novel VEGF or VEGFR orthologs in model organisms such as Drosophila melanogaster would provide tools for genetic and molecular study and validation of these molecules as potential pharmaceutical targets.
It is an object of the present invention to provide invertebrate homologs of VEGFR that can be used in genetic screening methods to characterize pathways that VEGFR may be involved in as well as other interacting genetic pathways. It is also an object of the invention to provide methods for screening compounds that interact with VEGFR such as those that may have utility as therapeutics.
These and other objects are provided by the present invention which concerns the identification and characterization of a novel VEGFR in Drosophila melanogaster, hereinafter referred to as xe2x80x9cdmVEGFRxe2x80x9d. Isolated nucleic acid molecules are provided that comprise nucleic acid sequences encoding dmVEGFR proteins as well as novel fragments and derivatives thereof. Methods of using the isolated nucleic acid molecules and fragments of the invention are described, such as use of RNA interference methods that block dmVEGFR activity. Vectors and host cells comprising the dmVEGFR nucleic acid molecules are also described, as well as metazoan invertebrate organisms (e.g. insects, coelomates and pseudocoelomates) that are genetically modified to express or mis-express a dmVEGFR protein.
An important utility of the novel dmVEGFR nucleic acids and proteins is that they can be used in screening assays to identify candidate compounds which are potential therapeutics that interact with dmVEGFR proteins. Further, the extracellular domain of dmVEGFR can be used as a reagent for purification of its ligand, as a biological probe of VEGF function in model organisms, and as an antagonist to VEGFR activity in vitro and in vivo.
Screening assays typically comprise contacting a dmVEGFR protein or fragment with one or more candidate molecules, and detecting any interaction between the candidate compound and the dmVEGFR protein. The assays may comprise administering the candidate molecules to cultured host cells that have been genetically engineered to express the dmVEGFR proteins, or alternatively, administering the candidate compound to a metazoan invertebrate organism that are genetically engineered to express a dmVEGFR protein.
The genetically engineered metazoan invertebrate animals of the invention can also be used in methods for studying dmVEGFR activity. These methods typically involve detecting the phenotype caused by the expression or mis-expression of the dmVEGFR protein. The methods may additionally comprise observing a second animal that has the same genetic modification as the first animal and, additionally has a mutation in a gene of interest. Any difference between the phenotypes of the two animals identifies the gene of interest as capable of modifying the function of the gene encoding the dmVEGFR protein.
The use of invertebrate model organism genetics and related technologies can greatly facilitate the elucidation of biological pathways (Scangos, Nat. Biotechnol. (1997) 15:1220-1221; Margolis and Duyk, supra). Of particular use is the insect model organism, Drosophila melanogaster (hereinafter referred to generally as xe2x80x9cDrosophilaxe2x80x9d). An extensive search for Vascular Endothelial Growth Factor Receptor, hereinafter referred to as VEGFR, nucleic acids and their encoded proteins in Drosophila was conducted in an attempt to identify new and useful tools for probing the function and regulation of the VEGFR genes, and for use as targets in drug discovery.
Novel VEGFR nucleic acids and their encoded proteins, hereinafter referred to as dmVEGFR, are identified herein. The newly identified dmVEGFR nucleic acids can be used for the generation of mutant phenotypes in animal models or in living cells that can be used to study regulation of dmVEGFR, and the use of dmVEGFR as a drug target. Due to the ability to rapidly carry out large-scale, systematic genetic screens, the use of invertebrate model organisms such as Drosophila has great utility for analyzing the expression and mis-expression of dmVEGFR protein. Thus, the invention provides a superior approach for identifying other components involved in the synthesis, activity, and regulation of dmVEGFR proteins. Systematic genetic analysis of VEGFRs using invertebrate model organisms can lead to the identification and validation of pesticide targets directed to components of the VEGFR pathway. Model organisms or cultured cells that have been genetically engineered to express dmVEGFR can be used to screen candidate compounds for their ability to modulate dmVEGFR expression or activity, and thus are useful in the identification of new drug targets, therapeutic agents, diagnostics and prognostics useful in the treatment of disorders associated with receptor tyrosine kinases. Additionally, these invertebrate model organisms can be used for the identification and screening of pesticide targets directed to components of the VEGFR pathway.
The details of the conditions used for the identification and/or isolation of novel dmVEGFR nucleic acids and proteins are described in the Examples section below. Various non-limiting embodiments of the invention, applications and uses of these novel dmVEGFR genes and proteins are discussed in the following sections. The entire contents of all references, including patent applications, cited herein are incorporated by reference in their entireties for all purposes. Additionally, the citation of a reference in the preceding background section is not an admission of prior art against the claims appended hereto.
The invention relates generally to nucleic acid sequences of VEGFRs, and more particularly VEGFR nucleic acid sequences of Drosophila, and methods of using these sequences. As described in the Examples below, nucleic acid sequences (SEQ ID NOs:1, 3, 5, and 7) were isolated from Drosophila that encode a VEGFR homologue. In addition to the fragments and derivatives of SEQ ID NOs:1, 3, 5, and 7 as described in detail below, the invention includes the reverse complements thereof. Also, the subject nucleic acid sequences, derivatives and fragments thereof may be RNA molecules comprising the nucleotide sequence of SEQ ID NOs:1, 3, 5, and 7 (or derivative or fragment thereof) wherein the base U (uracil) is substituted for the base T (thymine). The DNA and RNA sequences of the invention can be single- or double-stranded. Thus, the term xe2x80x9cisolated nucleic acid sequencexe2x80x9d, as used herein, includes the reverse complement, RNA equivalent, DNA or RNA single- or double-stranded sequences, and DNA/RNA hybrids of the sequence being described, unless otherwise indicated.
Fragments of the dmVEGFR nucleic acid sequences can be used for a variety of purposes. Interfering RNA (RNAi) fragments, particularly double-stranded (ds) RNAi, can be used to generate loss-of-function phenotypes, or to formulate biopesticides. dmVEGFR nucleic acid fragments are also useful as nucleic acid hybridization probes and replication/amplification primers. Certain xe2x80x9cantisensexe2x80x9d fragments, i.e. that are reverse complements of portions of the coding sequence of SEQ ID NOs:1, 3, 5, or 7 have utility in inhibiting the function of dmVEGFR proteins. The fragments are of length sufficient to specifically hybridize with the corresponding SEQ ID NOs:1, 3, 5, or 7. The fragments consist of or comprise at least 12, preferably at least 24, more preferably at least 36, and most preferably at least 96 contiguous nucleotides of SEQ ID NOs:1, 3, 5, or 7. In some cases, the fragments consist of or comprise at least 200, or more preferably at least 500 contiguous nucleotides of SEQ ID NOs:1, 3, 5, or 7. In-one preferred embodiment, the fragments of at least 12, 24, 36, 96, 200, 500, 995, or 1000 nucleotides are of any contiguous stretch of nucleotides 1 to 4040 of SEQ ID NOs:1, 3, 5, or 7. When these fragments are flanked by other nucleic acid sequences, the total length of the combined nucleic acid sequence is less than 20 kb, preferably less than 15 kb, 10 kb, or 5 kb, more preferably less than 2 kb, and in some cases, preferably less than 500 bases.
Additional preferred fragments of SEQ ID NO:1 encode domains having homology with immunoglobulin superfamily (IgSF) domains, which are located at approximately nucleotides 439-664, 820-1027, 1133-1346, 1441-1654 1748-1994, 2114-2309, and 1435-1609; extracellular domain, located at approximately nucleotides 368-2675; extracellular and transmembrane domain, located at approximately nucleotides 368-2744; and tyrosine kinase domains, located at approximately nucleotides 2906-3215, and 3620-4076.
Additional preferred fragments of SEQ ID NO:3 encode domains having homology with immunoglobulin superfamily (IgSF) domains, which are located at approximately nucleotides 228-357, 522-687, 828-1041, 1164-1338, 1443-1512, 1809-2004, and 2130-2304; extracellular domain, located at approximately nucleotides 1-2376; and tyrosine kinase domains, located at approximately nucleotides 2601-2910, and 3210-3666.
Additional preferred fragments of SEQ ID NO:5 encode domains having homology with immunoglobulin superfamily (IgSF) domains, which are located at approximately nucleotides 228-357, 522-687, 810-1023, 1146-1320, 1425-1494, 1791-1986, 2112-2286; extracellular domain, located at approximately nucleotides 1-2358; and tyrosine kinase domains, located at approximately nucleotides 2583-2892, and 3297-3753.
Additional preferred fragments of SEQ ID NO:7 encode domains having homology with immunoglobulin superfamily (IgSF) domains, which are located at approximately nucleotides 228-357, and 522-687; and extracellular domain, located at approximately nucleotides 1-759.
Other preferred fragments of SEQ ID NO:1 consist of or comprise a sequence of less than 15kb that encodes a stretch of at least 12, preferably at least 15, more preferably at least 20, and most preferably at least 25 contiguous amino acids from any of amino acids 1 to 1000 of SEQ ID NO:2 or amino acids 22 to 796 of SEQ ID NO:2. Other preferred fragments of SEQ ID NO:3 consist of or comprise a sequence of less than 15kb that encodes a stretch of at least 12, preferably at least 15, more preferably at least 20, and most preferably at least 25 contiguous amino acids from any of amino acids 1 to 1000 of SEQ ID NO:4 or amino acids 1-792 of SEQ ID NO:4.
Other preferred fragments of SEQ ID NO:5 consist of or comprise a sequence of less than 15kb that encodes a stretch of at least 12, preferably at least 15, more preferably at least 20, and most preferably at least 25 contiguous amino acids from any of amino acids 1 to 1000 of SEQ ID NO:6 or amino acids 1 to 786 of SEQ ID NO:6.
Other preferred fragments of SEQ ID NO:7 consist of or comprise a sequence of less than 15kb that encodes a stretch of at least 12, preferably at least 15, more preferably at least 20, and most preferably at least 25 contiguous amino acids from any of amino acids 1 to 253 of SEQ ID NO:8.
The subject nucleic acid sequences may consist solely of SEQ ID NOs:1, 3, 5, or 7 or fragments thereof. Alternatively, the subject nucleic acid sequences and fragments thereof may be joined to other components such as labels, peptides, agents that facilitate transport across cell membranes, hybridization-triggered cleavage agents or intercalating agents. The subject nucleic acid sequences and fragments thereof may also be joined to other nucleic acid sequences (i.e. they may comprise part of larger sequences) and are of synthetic/non-natural sequences and/or are isolated and/or are purified, i.e. unaccompanied by at least some of the material with which it is associated in its natural state. Preferably, the isolated nucleic acids constitute at least about 0.5%, and more preferably at least about 5% by weight of the total nucleic acid present in a given fraction, and are preferably recombinant, meaning that they comprise a non-natural sequence or a natural sequence joined to nucleotide(s) other than that which it is joined to on a natural chromosome.
Derivative nucleic acid sequences of dmVEGFR include sequences that hybridize to the nucleic acid sequence of SEQ ID NO:1 under stringency conditions such that the hybridizing derivative nucleic acid is related to the subject nucleic acid by a certain degree of sequence identity. A nucleic acid molecule is xe2x80x9chybridizablexe2x80x9d to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule. Stringency of hybridization refers to conditions under which nucleic acids are hybridizable. The degree of stringency can be controlled by temperature, ionic strength, pH, and the presence of denaturing agents such as formamide during hybridization and washing. As used herein, the term xe2x80x9cstringent hybridization conditionsxe2x80x9d are those normally used by one of skill in the art to establish at least a 90% sequence identity between complementary pieces of DNA or DNA and RNA. xe2x80x9cModerately stringent hybridization conditionsxe2x80x9d are used to find derivatives having at least 70% sequence identity. Finally, xe2x80x9clow-stringency hybridization conditionsxe2x80x9d are used to isolate derivative nucleic acid molecules that share at least about 50% sequence identity with the subject-nucleic acid sequence.
The ultimate hybridization stringency reflects both the actual hybridization conditions as well as the washing conditions following the hybridization, and it is well known in the art how to vary the conditions to obtain the desired result. Conditions routinely used are set out in readily available procedure texts (e.g., Current Protocol in Molecular Biology, Vol. 1, Chap. 2.10, John Wiley and Sons, Publishers (1994); Sambrook et al., Molecular Cloning, Cold Spring Harbor (1989)). A preferred derivative nucleic acid is capable of hybridizing to SEQ ID NO:1 under stringent hybridization conditions that comprise: prehybridization of filters containing nucleic acid for 8 hours to overnight at 65xc2x0 C. in a solution comprising 6xc3x97single strength citrate (SSC) (1xc3x97SSC is 0.15 M NaCl, 0.015 M Na citrate; pH 7.0), 5xc3x97Denhardt""s solution, 0.05% sodium pyrophosphate and 100 xcexcg/ml herring sperm DNA; hybridization for 18-20 hours at 65xc2x0 C. in a solution containing 6xc3x97SSC, 1xc3x97Denhardt""s solution, 100 xcexcg/ml yeast tRNA and 0.05% sodium pyrophosphate; and washing of filters at 65xc2x0 C. for 1 h in a solution containing 0.2xc3x97SSC and 0.1% SDS (sodium dodecyl sulfate).
Derivative nucleic acid sequences that have at least about 70% sequence identity with any of SEQ ID NOs:1, 3, 5, and 7 are capable of hybridizing to SEQ ID NOs:1, 3, 5, or 7 under moderately stringent conditions that comprise: pretreatment of filters containing nucleic acid for 6 h at 40xc2x0 C. in a solution containing 35% formamide, 5xc3x97SSC, 50 mM Tris-HCl (pH7.5), 5 mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 xcexcg/ml denatured salmon sperm DNA; hybridization for 18-20 h at 40xc2x0 C. in a solution containing 35% formamide, 5xc3x97SSC, 50 mM Tris-HCl (pH7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 xcexcg/ml salmon sperm DNA, and 10% (wt/vol) dextran sulfate; followed by washing twice for 1 hour at 55xc2x0 C. in a solution containing 2xc3x97SSC and 0.1% SDS.
Other preferred derivative nucleic acid sequences are capable of hybridizing to any of SEQ ID NOs:1, 3, 5, or 7 under low stringency conditions that comprise: incubation for 8 hours to overight at 37xc2x0 C. in a solution comprising 20% formamide, 5xc3x97SSC, 50 mM sodium phosphate (pH 7.6), 5xc3x97Denhardt""s solution, 10% dextran sulfate, and 20 xcexcg/ml denatured sheared salmon sperm DNA; hybridization in the same buffer for 18 to 20 hours; and washing of filters in 1xc3x97SSC at about 37xc2x0 C. for 1 hour.
As used herein, xe2x80x9cpercent (%) nucleic acid sequence identityxe2x80x9d with respect to a subject sequence, or a specified portion of a subject sequence, is defined as the percentage of nucleotides in the candidate derivative nucleic acid sequence identical with the nucleotides in the subject sequence (or specified portion thereof), after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent sequence identity, as generated by the program WU-BLAST-2.0a19 (Altschul et al., J. Mol. Biol. (1997) 215:403-410; hereinafter referred to generally as xe2x80x9cBLASTxe2x80x9d) with all the search parameters set to default values. The HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched. A percent (%) nucleic acid sequence identity value is determined by the number of matching identical nucleotides divided by the sequence length for which the percent identity is being reported.
Derivative dmVEGFR nucleic acid sequences usually have at least 70% sequence identity, preferably at least 80% sequence identity, more preferably at least 85% sequence identity, still more preferably at least 90% sequence identity, and most preferably at least 95% sequence identity with any of SEQ ID NOs:1, 3, 5, and 7 or a domain-encoding region thereof. In one preferred embodiment, the derivative nucleic acid encodes a polypeptide comprising a dmVEGFR amino acid sequence of any of SEQ ID NOs:2, 4, 6, 8 or a fragment or derivative thereof as described further below under the subheading xe2x80x9cdmVEGFR proteinsxe2x80x9d. In a preferred embodiment, a derivative of any of SEQ ID NOs:1, 3, 5, and 7 consists of or comprises a sequence of less than 15kb that encodes a stretch of at least 20, preferably 22, more preferably 25, and most preferably at least 30 contiguous amino acids that share 100% sequence similarity with an equivalent number of contiguous amino acids from any of amino acids 1 to 796 of any of SEQ ID NOs:2, 4, or 6 or amino acids 1 to 253 of SEQ ID NO:8.
A derivative dmVEGFR nucleic acid sequence, or fragment thereof, may comprise 100% sequence identity with any of SEQ ID NOs:1, 3, 5, or 7 but be a derivative thereof in the sense that it has one or more modifications at the base or sugar moiety, or phosphate backbone. Examples of modifications are well known in the art (Bailey, Ullmann""s Encyclopedia of Industrial Chemistry (1998), 6th ed. Wiley and Sons). Such derivatives may be used to provide modified stability or any other desired property.
Another type of derivative of the subject nucleic acid sequences includes corresponding humanized sequences. A humanized nucleic acid sequence is one in which one or more codons has been substituted with a codon that is more commonly used in human genes. Preferably, a sufficient number of codons have been substituted such that a higher level expression is achieved in mammalian cells than what would otherwise be achieved without the substitutions. The following list shows, for each amino acid, the calculated codon frequency (number in parentheses) in humans genes for 1000 codons (Wada et al., Nucleic Acids Research (1990) 18(Suppl.):2367-2411):
Human codon frequency per 1000 codons:
ARG: CGA (5.4), CGC (11.3), CGG (10.4), CGU (4.7), AGA (9.9), AGG (11.1)
LEU: CUA (6.2), CUC (19.9), CUG (42.5), CUU (10.7), UUA (5.3), UUG (11.0)
SER: UCA (9.3), UCC (17.7), UCG (4.2), UCU (13.2), AGC (18.7), AGU (9.4)
THR: ACA (14.4), ACC (23.0), ACG (6.7), ACU (12.7)
PRO: CCA (14.6), CCC (20.0), CCG (6.6), CCU (15.5)
ALA: GCA (14.0), GCC (29.1), GCG (7.2), GCU (19.6)
GLY: GGA (17.1), GGC (25.4), GGG (17.3), GGU (11.2)
VAL: GUA (5.9), GUC (16.3), GUG (30.9), GUU (10.4)
LYS: AAA (22.2), AAG (34.9)
ASN: AAC (22.6), AAU (16.6)
GLN: CAA (11.1), CAG (33.6)
HIS: CAC (14.2), CAU (9.3)
GLU: GAA (26.8), GAG (41.4)
ASP: GAC (29.0), GAU (21.7)
TYR: UAC (18.8), UAU (12.5)
CYS: UGC (14.5), UGU (9.9)
PHE: UUU (22.6), UUC (15.8)
ILE: AUA (5.8), AUC (24.3), AUU (14.9)
MET: AUG (22.3)
TRP: UGG (13.8)
TER: UAA (0.7), AUG (0.5), UGA (1.2)
Thus, a dmVEGFR nucleic acid sequence in which the glutamic acid codon, GAA has been replaced with the codon GAG, which is more commonly used in human genes, is an example of a humanized dmVEGFR nucleic acid sequence. A detailed discussion of the humanization of nucleic acid sequences is provided in U.S. Pat. No. 5,874,304 to Zolotukhin et al.
Nucleic acids encoding the amino acid sequence of any of SEQ ID NOs:2, 4, 6, or 8 or fragments or derivatives thereof, may be obtained from an appropriate cDNA library prepared from any eukaryotic species that encodes VEGFR proteins such as vertebrates, preferably mammalian (e.g. primate, porcine, bovine, feline, equine, and canine species, etc.) and invertebrates, such as arthropods, particularly insects species (preferably Drosophila), acarids, crustacea, molluscs, nematodes, and other worms. An expression library can be constructed using known methods. For example, mRNA can be isolated to make cDNA which is ligated into a suitable expression vector for expression in a host cell into which it is introduced. Various screening assays can.then be used to select for the gene or gene product (e.g. oligonucleotides of at least about 20 to 80 bases designed to identify the gene of interest, or labeled antibodies that specifically bind to the gene product). The gene and/or gene product can then be recovered from the host cell using known techniques.
Polymerase chain reaction (PCR) can also be used to isolate nucleic acids of the dmVEGFR where oligonucleotide primers representing fragmentary sequences of interest amplify RNA or DNA sequences from a source such as a genomic or cDNA library (as described by Sambrook et al., supra). Additionally, degenerate primers for amplifying homologues from any species of interest may be used. Once a PCR product of appropriate size and sequence is obtained, it may be cloned and sequenced by standard techniques, and utilized as a probe to isolate a complete cDNA or genomic clone.
Fragmentary sequences of dmVEGFR nucleic acids and derivatives may be synthesized by known methods. For example, oligonucleotides may be synthesized using an automated DNA synthesizer available from commercial suppliers (e.g. Biosearch, Novato, Calif.; Perkin-Elmer Applied Biosystems, Foster City, Calif.). Antisense RNA sequences can be produced intracellularly by transcription from an exogenous sequence, e.g. from vectors that contain antisense dmVEGFR nucleic acid sequences. Newly generated sequences may be identified and isolated using standard methods.
An isolated dmVEGFR nucleic acid sequence can be inserted into any appropriate cloning vector, for example bacteriophages such as lambda derivatives, or plasmids such as PBR322, pUC plasmid derivatives and the Bluescript vector (Stratagene, San Diego, Calif.). Recombinant molecules can be introduced into host cells via transformation, transfection, infection, electroporation, etc., or into a transgenic animal such as a fly. The transformed cells can be cultured to generate large quantities of the dmVEGFR nucleic acid. Suitable methods for isolating and producing the subject nucleic acid sequences are well-known in the art (Sambrook et al., supra; DNA Cloning: A Practical Approach, Vol. 1, 2, 3, 4, (1995) Glover, ed., MRL Press, Ltd., Oxford, U.K.).
The nucleotide sequence encoding a dmVEGFR protein or fragment or derivative thereof, can be inserted into any appropriate expression vector for the transcription and translation of the inserted protein-coding sequence. Alternatively, the necessary transcriptional and translational signals can be supplied by the native dmVEGFR gene and/or its flanking regions. A variety of host-vector systems may be utilized to express the protein-coding sequence such as mammalian cell systems infected with virus (e.g. vaccinia virus, adenoviruts, etc.); insect cell systems infected with virus (e.g. baculovirus); microorganisms such as yeast containing yeast vectors, or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. Expression of a dmVEGFR protein may be controlled by a suitable promoter/enhancer element. In addition, a host cell strain may be selected which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired.
To detect expression of the dmVEGFR gene product, the expression vector can comprise a promoter operably linked to a dmVEGFR gene nucleic acid, one or more origins of replication, and, one or more selectable markers (e.g. thymidine kinase activity, resistance to antibiotics, etc.). Alternatively, recombinant expression vectors can be identified by assaying for the expression of the dmVEGFR gene product based on the physical or functional properties of the dmVEGFR protein in in vitro assay systems (e.g. immunoassays).
The dmVEGFR protein, fragment, or derivative may be optionally expressed as a fusion, or chimeric protein product (i.e. it is joined via a peptide bond to a heterologous protein sequence of a different protein). A chimeric product can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other in the proper coding frame using standard methods and expressing the chimeric product. A chimeric product may also be made by protein synthetic techniques, e.g. by use of a peptide synthesizer.
Once a recombinant that expresses the dmVEGFR gene sequence is identified, the gene product can be isolated and purified using standard methods (e.g. ion exchange, affinity, and gel exclusion chromatography; centrifugation; differential solubility; electrophoresis). The amino acid sequence of the protein can be deduced from the nucleotide sequence of the chimeric gene contained in the recombinant and can thus be synthesized by standard chemical methods (Hunkapiller et al., Nature (1984) 310:105-111). Alternatively, native dmVEGFR proteins can be purified from natural sources, by standard methods (e.g. immunoaffinity purification).
dmVEGFR proteins of the invention comprise or consist of an amino acid sequence of SEQ ID NOs:2, 4,6, and 8 or fragments or derivatives thereof. Compositions comprising these proteins may consist essentially of the dmVEGFR protein, fragments, or derivatives, or may comprise additional components (e.g. pharmaceutically acceptable carriers or excipients, culture media, carriers used in pesticide formulations, etc.).
dmVEGFR protein derivatives typically share a certain degree of sequence identity or sequence similarity with any of SEQ ID NOs:2, 4, 6, or 8, or a fragment thereof. As used herein, xe2x80x9cpercent (%) amino acid sequence identityxe2x80x9d with respect to a subject sequence, or a specified portion of a subject sequence, is defined as the percentage of amino acids in the candidate derivative amino acid sequence identical with the amino acid in the subject sequence (or specified portion thereof), after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent sequence identity, as generated by BLAST (Altschul et al., supra) using the same parameters discussed above for derivative nucleic acid sequences. A % amino acid sequence identity value is determined by the number of matching identical amino acids divided by the sequence length for which the percent identity is being reported. xe2x80x9cPercent (%) amino acid sequence similarityxe2x80x9d is determined by doing the same calculation as for determining % amino acid sequence identity, but including conservative amino acid substitutions in addition to identical amino acids in the computation. A conservative amino acid substitution is one in which an amino acid is substituted for another amino acid having similar properties such that the folding or activity of the protein is not significantly affected. Aromatic amino acids that can be substituted for each other are phenylalanine, tryptophan, and tyrosine; interchangeable hydrophobic amino acids are leucine, isoleucine, methionine, and valine; interchangeable polar amino acids are glutamine and asparagine; interchangeable basic amino acids are arginine, lysine and histidine; interchangeable acidic amino acids are aspartic acid and glutamic acid; and interchangeable small amino acids are alanine, serine, threonine, and glycine.
In one preferred embodiment, a dmVEGFR protein derivative shares at least 80% sequence identity or similarity, preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity or similarity with a contiguous stretch of at least 20 amino acids, preferably at least 25 amino acids, more preferably at least 30 amino acids, and in some cases, the entire length of SEQ ID NOs:2, 4, 6, or 8. In a preferred embodiment, the stretch of at least 20, 25, or 30 contiguous amino acids is from any of amino acids 1 to 1000 of SEQ ID NOs:2, 4, or 6, any amino acids 22 to 796 of SEQ ID NOs:2, 4, 6, or any amino acids 1-253 of SEQ ID NO:8.
In another embodiment, the dmVEGFR protein derivative may consist of or comprise a sequence that shares 100% similarity with any contiguous stretch of at least 20 amino acids, preferably at least 22 amino acids, more preferably at least 25 amino acids, and most preferably at least 28 amino acids of any of SEQ ID NOs:2, 4, 6, and 8, or any of amino acids 1 to 1000 of SEQ ID NOs:2, 4, and 6, or any of amino acids 22 to 796 of any of SEQ ID NOs:2, 4, or 6.
Preferred derivatives of dmVEGFR consist of or comprise an amino acid sequence that has at least 70%, preferably at least 80%, more preferably at least 85%, still more preferably at least 90%, and most preferably at least 95% sequence identity or sequence similarity with any of amino acid residues 22-796, which is the likely extracellular domain; residues 22-813, the extracellular and transmembrane domains; residues 45-120, 172-241, 276-347, 379-450, 481-563, 603-668, and 709-768, the immunoglobulin homology (IgG-like) domains; residues 867-970, and 1105-1257, the tyrosine kinase domains; or the entire length of SEQ ID NO:2.
Other preferred derivatives of dmVEGFR consist of or comprise an amino acid sequence that has at least 70%, preferably at least 80%, more preferably at least 85%, still more preferably at least 90%, and most preferably at least 95% sequence identity or sequence similarity with any of amino acid residues 22-796, which is the likely extracellular domain; residues 76-119, 174-229, 276-347, 388-446, 481-504, 603-668, and 710-768, the immunoglobulin homology (IgG-like) domains; residues 867-970, and 1070-1222, the tyrosine kinase domains; or the entire length of SEQ ID NO:4.
Further preferred derivatives of dmVEGFR consist of or comprise an amino acid sequence that has at least 70%, preferably at least 80%, more preferably at least 85%, still more preferably at least 90%, and most preferably at least 95% sequence identity or sequence similarity with any of amino acid residues 22-786, which is the likely extracellular domain; residues 76-119, 174-229, 270-341, 382-440, 475-498, 597-662, and 704-762, the immunoglobulin homology (IgG-like) domains; residues 861-964, and 1099-1251, the tyrosine kinase domains; or the entire length of SEQ ID NO:6.
Still further preferred derivatives of dmVEGFR consist of or comprise an amino acid sequence that has at least 70%, preferably at least 80%, more preferably at least 85%, still more preferably at least 90%, and most preferably at least 95% sequence identity or sequence similarity with any of amino acid residues 22-253, which is the likely extracellular domain, or the entire length of SEQ ID NO:8.
Preferred fragments of dmVEGFR proteins consist or comprise at least 12, preferably at least 14, more preferably at least 17, and most preferably at least 22 contiguous amino acids of any of SEQ ID NOs:2, 4, 6, or 8, any of amino acids. 1 to 1000 of SEQ ID NOs:2, 4, or 6, any of amino acids 22 to 796 of SEQ ID NOs:2, 4, 6, or the entire length of SEQ ID NO:8. The fragment or derivative of the dmVEGFR protein is preferably xe2x80x9cfunctionally activexe2x80x9d meaning that the dmVEGFR protein derivative or fragment exhibits one or more functional activities associated with a full-length, wild-type VEGFR protein comprising the-amino acid sequence of SEQ ID NOs:2, 4, 6, or 8. As one example, a fragment or derivative may have antigenicity such that it can be used in immunoassays, for immunization, for inhibition of dmVEGFR activity, etc, as discussed further below regarding generation of antibodies to dmVEGFR proteins. Preferably, a functionally active dmVEGFR fragment or derivative is one that displays one or more biological activities associated with VEGFR proteins such as tyrosine kinase receptor activity. For purposes herein, functionally active fragments also include those fragments that exhibit one or more structural features of a VEGFR, such as the extracellular domain including the immunoglobulin homology domains, the transmembrane domain, and the intracellular domain containing the tyrosine kinase domains. The functional activity of dmVEGFR proteins, derivatives and fragments can be assayed by various methods known to one skilled in the art (Current Protocols in Protein Science (1998) Coligan et al., eds., John Wiley and Sons, Inc., Somerset, N.J.). In a preferred method, which is described in detail below, a model organism, such as Drosophila, is used in genetic studies to assess the phenotypic effect of a fragment or derivative (i.e. a mutant dmVEGFR protein).
dmVEGFR derivatives can be produced by various methods known in the art. The manipulations which result in their production can occur at the gene or protein level. For example, a cloned VEGFR gene sequence can be cleaved at appropriate sites with restriction endonuclease(s) (Wells et al., Philos. Trans. R. Soc. London SerA (1986) 317:415), followed by further enzymatic modification if desired, isolated, and ligated in vitro, and expressed to produce the desired derivative. Alternatively, a VEGFR gene can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or to form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification. A variety of mutagenesis techniques are known in the art such as chemical mutagenesis, in vitro site-directed mutagenesis (Carter et al., Nucl. Acids Res. (1986) 13:4331), use of TAB(copyright) linkers (available from Pharmacia and Upjohn, Kalamazoo, Mich.), etc.
At the protein level, manipulations include post translational modification, e.g. glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known technique (e.g. specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH4, acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc.). Derivative proteins can also be chemically synthesized by use of a peptide synthesizer, for example to introduce nonclassical amino acids or chemical amino acid analogs as substitutions or additions into the dmVEGFR protein sequence.
Chimeric or fusion proteins can be made comprising a VEGFR protein or fragment thereof (preferably comprising one or more structural or functional domains of the dmVEGFR protein) joined at its amino- or carboxy-terminus via a peptide bond to an amino acid sequence of a different protein. Chimeric proteins can be produced by any known method, including: recombinant expression of a nucleic acid encoding the protein (comprising a VEGFR-coding sequence joined in-frame to a coding sequence for a different protein); ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other in the proper coding frame, and expressing the chimeric product; and protein synthetic techniques, e.g. by use of a peptide synthesizer.
dmVEGFR gene regulatory DNA elements such as enhancers or promoters that reside within nucleotides 1 to 304 of SEQ ID NO:1, can be used to identify tissues, cells, genes and factors that specifically control dmVEGFR protein production. Preferably least 20, more preferably at least 25, and most preferably at least 50 contiguous nucleotides within nucleotides 1 to 304 of SEQ ID NO:1 are used. Analyzing components that are specific to dmVEGFR protein function can lead to an understanding of how to manipulate these regulatory processes, especially for pesticide and therapeutic applications, as well as an understanding of how to diagnose dysfunction in these processes.
Gene fusions with the dmVEGFR regulatory elements can be made. For compact genes that have relatively few and small intervening sequences, such as those described herein for Drosophila, it is typically the case that the regulatory elements that control spatial and temporal expression patterns are found in the DNA immediately upstream of the coding region, extending to the nearest neighboring gene. Regulatory regions can be used to construct gene fusions where the regulatory DNAs are operably fused to a coding region for a reporter protein whose expression is easily detected, and these constructs are introduced as transgenes into the animal of choice. An entire regulatory DNA region can be used, or the regulatory region can be divided into smaller segments to identify sub-elements that might be specific for controlling expression a given cell type or stage of development. Reporter proteins that can be used for construction of these gene fusions include E. coli beta-galactosidase and green fluorescent protein (GFP). These can be detected readily in situ, and thus are useful for histological studies and can be used to sort cells that express dmVEGFR proteins (OKane and Gehring PNAS (1987) 84(24):9123-9127; Chalfie et al., Science (1994) 263:802-805; and Cumberledge and Krasnow (1994) Methods in Cell Biology 44:143-159). Recombinase proteins, such as FLP or Cre, can be used in controlling gene expression through site-specific recombination (Golic and Lindquist (1989) Cell 59(3):499-509; White et al., Science (1996) 271:805-807). Toxic proteins such as the reaper and hid cell death proteins, are useful to specifically ablate cells that normally express dmVEGFR proteins in order to assess the physiological function of the cells (Kingston, In Current Protocols in Molecular Biology (1998) Ausubel et al., John Wiley and Sons, Inc. sections 12.0.3-12.10) or any other protein where it is desired to examine the function this particular protein specifically in cells that synthesize dmVEGFR proteins.
Alternatively, a binary reporter system can be used, similar to that described further below, where the dmVEGFR regulatory element is operably fused to the coding region of an exogenous transcriptional activator protein, such as the GAL4 or tTA activators described below, to create a dmVEGFR regulatory element xe2x80x9cdriver genexe2x80x9d. For the other half of the binary system the exogenous activator controls a separate xe2x80x9ctarget genexe2x80x9d containing a coding region of a reporter protein operably fused to a cognate regulatory element for the exogenous activator protein, such as UASG or a tTA-response element, respectively. An advantage of a binary system is that a single driver gene construct can be used to activate transcription from preconstructed target genes encoding different reporter proteins, each with its own uses as delineated above.
dmVEGFR regulatory element-reporter gene fusions are also useful for tests of genetic interactions, where the objective is to identify those genes that have a specific role in controlling the expression of dmVEGFR genes, or promoting the growth and differentiation of the tissues that expresses the dmVEGFR proteins. dmVEGFR gene regulatory DNA elements are also useful in protein-DNA binding assays to identify gene regulatory proteins that control the expression of dmVEGFR genes. The gene regulatory proteins can be detected using a variety of methods that probe specific protein-DNA interactions well known to those skilled in the art (Kingston, supra) including in vivo footprinting assays based on protection of DNA sequences from chemical and enzymatic modification within living or permeabilized cells; and in vitro footprinting assays based on protection of DNA sequences from chemical or enzymatic modification using protein extracts, nitrocellulose filter-binding assays and gel electrophoresis mobility shift assays using radioactively labeled regulatory DNA elements mixed with protein extracts. Candidate dmVEGFR gene regulatory proteins can be purified using a combination of conventional and DNA-affinity purification techniques. Molecular cloning strategies can also be used to identify proteins that specifically bind dmVEGFR gene regulatory DNA elements. For example, a Drosophila cDNA library in an expression vector, can be screened for cDNAs that encode dmVEGFR gene regulatory element DNA-binding activity. Similarly, the yeast xe2x80x9cone-hybridxe2x80x9d system can be used (Li and Herskowitz, Science (1993) 262:1870-1874; Luo et al., Biotechniques (1996) 20(4):564-568; Vidal et al., PNAS (1996) 93(19):10315-10320).
A variety of methods can be used to identify or screen for molecules, such as proteins or other molecules, that interact with dmVEGFR proteins, or derivatives or fragments thereof. The assays may employ purified dmVEGFR protein, or cell lines or model organisms such as Drosophila and C. elegans, that have been genetically engineered to express dmVEGFR protein. Suitable screening methodologies are well known in the art to test for proteins and oiher molecules that interact with dmVEGFR genes and proteins (see e.g. PCT International Publication No. WO 96/34099). For example, the extracellular domain of dmVEGFR can be labeled and used as a probe to isolate VEGFR ligands. The newly identified interacting molecules may provide new targets for pharmaceutical agents. Any of a variety of exogenous molecules, both naturally occurring and/or synthetic (e.g., libraries of small molecules or peptides, or phage display libraries), may be screened for binding capacity. In a typical binding experiment, the dmVEGFR protein or fragment is mixed with candidate molecules under conditions conducive to binding, sufficient time is allowed for any binding to occur, and assays are performed to test for bound complexes. Assays to find interacting proteins can be performed by any method known in the art, for example, immunoprecipitation with an antibody that binds to the protein in a complex followed by analysis by size fractionation of the immunoprecipitated proteins (e.g. by denaturing or nondenaturing polyacrylamide gel electrophoresis), Western analysis, non-denaturing gel electrophoresis, etc.
Two-hybrid Assay Systems
A preferred method for identifying interacting proteins is a two-hybrid assay system or variation thereof (Fields and Song, Nature (1989) 340:245-246; U.S. Pat. No. 5,283,173; for review see Brent and Finley, Annu. Rev. Genet. (1997) 31:663-704). The most commonly used two-hybrid screen system is performed using yeast. All systems share three elements: 1) a gene that directs the synthesis of a xe2x80x9cbaitxe2x80x9d protein fused to a DNA binding domain; 2) one or more xe2x80x9creporterxe2x80x9d genes having an upstream binding site for the bait, and 3) a gene that directs the synthesis of a xe2x80x9cpreyxe2x80x9d protein fused to an activation domain that activates transcription of the reporter gene. For the screening of proteins that interact with dmVEGFR protein, the xe2x80x9cbaitxe2x80x9d is preferably a dmVEGFR protein, expressed as a fusion protein to a DNA binding domain; and the xe2x80x9cpreyxe2x80x9d protein is a protein to be tested for ability to interact with the bait, and is expressed as a fusion protein to a transcription activation domain. The prey proteins can be obtained from recombinant biological libraries expressing random peptides.
The bait fusion protein can be constructed using any suitable DNA binding domain, such as the E. coli LexA repressor protein, or the yeast GAL4 protein (Bartel et al., BioTechniques (1993) 14:920-924, Chasman et al., Mol. Cell. Biol. (1989) 9:4746-4749; Ma et al., Cell (1987) 48:847-853; Ptashne et al., Nature (1990) 346:329-331).
The prey fusion protein can be constructed using any suitable activation domain such as GAM4, VP-16, etc. The preys may contain useful moieties such as nuclear localization signals (Ylikomi et al., EMBO J. (1992) 11:3681-3694; Dingwall and Laskey, Trends Biochem. Sci. Trends Biochem. Sci. (1991) 16:479481) or epitope tags (Allen et al., Trends Biochem. Sci. Trends Biochem. Sci. (1995) 20:511-516) to facilitate isolation of the encoded proteins.
Any reporter gene can be used that has a detectable phenotype such as reporter genes that allow cells expressing them to be selected by growth on appropriate medium (e.g. HIS3, LEU2 described by Chien et al., PNAS (1991) 88:9572-9582; and Gyuris et al., Cell (1993) 75:791-803). Other reporter genes, such as LacZ and GFP, allow cells expressing them to be visually screened (Chien et al., supra).
Although the preferred host for two-hybrid screening is the yeast, the host cell in which the interaction assay and transcription of the reporter gene occurs can be any cell, such as mammalian (e.g. monkey, mouse, rat, human, bovine), chicken, bacterial, or insect cells. Various vectors and host strains for expression of the two fusion protein populations in yeast can be used (U.S. Pat. No. 5,468,614; Bartel et al., Cellular Interactions in Development (1993) Hartley, ed., Practical Approach Series xviii, IRL Press at Oxford University Press, New York, N.Y., pp. 153-179; and Fields and Sternglanz, Trends In Genetics (1994) 10:286-292). As an example of a mammalian system, interaction of activation tagged VP16 derivatives with a GAL4-derived bait drives expression of reporters that direct the synthesis of hygromycin B phosphotransferase, chloramphenicol acetyltransferase, or CD4 cell surface antigen (Fearon et al., PNAS (1992) 89:7958-7962). As another example, interaction of VP16-tagged derivatives with GAL4-derived baits drives the synthesis of SV40 T antigen, which in turn promotes the replication of the prey plasmid, which carries an SV40 origin (Vasavada et al., PNAS (1991) 88:10686-10690).
Typically, the bait dmVEGFR gene and the prey library of chimeric genes are combined by mating the two yeast strains on solid or liquid media for a period of approximately 6-8 hours. The resulting diploids contain both kinds of chimeric genes, i.e., the DNA-binding domain fusion and the activation domain fusion.
Transcription of the reporter gene can be detected by a linked replication assay in the case of SV40 T antigen (described by Vasavada et al., supra) or using immunoassay methods, preferably as described in Alam and Cook (Anal. Biochem. (1990)188:245-254). The activation of other reporter genes like URA3, HIS3, LYS2, or LEU2 enables the cells to grow in the absence of uracil, histidine, lysine, or leucine, respectively, and hence serves as a selectable marker. Other types of reporters are monitored by measuring a detectable signal. For example, GFP and lacZ have gene products that are fluorescent and chromogenic, respectively. After interacting proteins have been identified, the DNA sequences encoding the proteins can be isolated. In one method, the activation domain sequences or DNA-binding domain sequences (depending on the prey hybrid used) are amplified, for example, by PCR using pairs of oligonucleotide primers specific for the coding region of the DNA binding domain or activation domain. Other known amplification methods can be used, such as ligase chain reaction, use of Q replicase, or various other methods described (see Kricka et al., Molecular Probing, Blotting, and Sequencing (1995) Academic Press, New York, Chapter 1 and Table IX).
If a shuttle (yeast to E. coli) vector is used to express the fusion proteins, the DNA sequences encoding the proteins can be isolated by transformation of E. coli using the yeast DNA and recovering the plasmids from E. coli. Alternatively, the yeast vector can be isolated, and the insert encoding the fusion protein subcloned into a bacterial expression vector, for growth of the plasmid in E. coli. 
A limitation of the two-hybrid system occurs when transmembrane portions of proteins in the bait or the prey fusions are used. This occurs because most two-hybrid systems are designed to function by formation of a functional transcription activator complex within the nucleus, and use of transmembrane portions of the protein can interfere with proper association, folding, and nuclear transport of bait or prey segments (Ausubel et al., supra; Allen et al., supra). Since the dmVEGFR protein is a transmembrane protein, it is preferred that intracellular or extracellular domains be used for bait in a two-hybrid scheme.
Antibodies to dmVEGFR and Immunoassays
dmVEGFR proteins encoded by any of SEQ ID NOs:2, 4, 6, or 8 and derivatives and fragments thereof, such as those discussed above, may be used as an immunogen to generate monoclonal or polyclonal antibodies and antibody fragments or derivatives (e.g. chimeric, single chain, Fab fragments). For example, fragments of a dmVEGFR protein, preferably those identified as hydrophilic, are used as immunogens for antibody production using art-known methods such as by hybridomas; production of monoclonal antibodies in germ-free animals (PCT/US90/02545); the use of human hybridomas (Cole et al., PNAS (1983) 80:2026-2030; Cole et al., in Monoclonal Antibodies and Cancer Therapy (1985) Alan R. Liss, pp. 77-96), and production of humanized antibodies (Jones et al., Nature (1986) 321:522-525; U.S. Pat. No. 5,530,101). In a particular embodiment, dmVEGFR polypeptide fragments provide specific antigens and/or immunogens, especially when coupled to carrier proteins. For example, peptides are covalently coupled to keyhole limpet antigen (KLH) and the conjugate is emulsified in Freund""s complete adjuvant. Laboratory rabbits are immunized according to conventional protocol and bled. The presence of specific antibodies is assayed by solid phase immunosorbent assays using immobilized corresponding polypeptide. Specific activity or function of the antibodies produced may be determined by convenient in vitro, cell-based, or in vivo assays: e.g. in vitro binding assays, etc. Binding affinity may be assayed by determination of equilibrium constants of antigen-antibody association (usually at least about 107M-, preferably at least about 108 Mxe2x88x921, more preferably at least about 109 Mxe2x88x921).
Immunoassays can be used to identify proteins that interact with or bind to dmVEGFR protein. Various assays are available for testing the ability of a protein to bind to or compete with binding to a wild-type dmVEGFR protein or for binding to an anti-dmVEGFR protein antibody. Suitable assays include radioimmunoassays, ELISA (enzyme linked immunosorbent assay), immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (e.g., using colloidal gold, enzyme or radioisotope labels), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, immunoelectrophoresis assays, etc.
VEGFR genes or interacting genes can be assessed as potential drug or pesticide targets. Putative drugs and molecules can be applied onto whole insects, nematodes, and other small invertebrate metazoans, and the ability of the compounds to modulate (e.g. block or enhance) dmVEGFR activity can be observed. Alternatively, the effect of various compounds on dmVEGFRs can be assayed using cells that have been engineered to express one or more dmVEGFRs and associated proteins.
Assays of Compounds on Worms
In a typical worm assay, the compounds to be tested are dissolved in DMSO or other organic solvent, mixed with a bacterial suspension at various test concentrations, preferably OP50 strain of bacteria (Brenner, Genetics (1974) 110:421-440), and supplied as food to the a worms. The population of worms to be treated can be synchronized larvae (Sulston and Hodgkin, in The nematode C. elegans (1988), supra) or adults or a mixed-stage population of animals.
Adult and larval worms are treated with different concentrations of compounds, typically ranging from 1 mg/ml to 0.001 mg/ml. Behavioral aberrations, such as a decrease in motility and growth, and morphological aberrations, sterility, and death are examined in both acutely and chronically treated adult and larval worms. For the acute assay, larval and adult worms are examined immediately after application of the compound and re-examined periodically (every 30 minutes) for 5-6 hours. Chronic or long-term assays are performed on worms and the behavior of the treated worms is examined every 8-12 hours for 4-5 days. In some circumstances, it is necessary to reapply the pesticide to the treated worms every 24 hours for maximal effect.
Assays of Compounds on Insects
Potential insecticidal compounds can be administered to insects in a variety of ways, including orally (including addition to synthetic diet, application to plants or prey to be consumed by the test organism), topically (including spraying, direct application of compound to animal, allowing animal to contact a treated surface), or by injection. Insecticides are typically very hydrophobic molecules and must commonly be dissolved in organic solvents, which are allowed to evaporate in the case of methanol or acetone, or at low concentrations can be included to facilitate uptake (ethanol, dimethyl sulfoxide).
The first step in an insect assay is usually the determination of the minimal lethal dose (MLD) on the insects after a chronic exposure to the compounds. The compounds are usually diluted in DMSO, and applied to the food surface bearing 0-48 hour old embryos and larvae. In addition to MLD, this step allows the determination of the fraction of eggs that hatch, behavior of the larvae, such as how they move/feed compared to untreated larvae, the fraction that survive to pupate, and the fraction that eclose (emergence of the adult insect from puparium). Based on these results more detailed assays with shorter exposure times may be designed, and larvae might be dissected to look for obvious morphological defects. Once the MLD is determined, more specific acute and chronic assays can be designed.
In a typical acute assay, compounds are applied to the food surface for embryos, larvae, or adults, and the animals are observed after 2 hours and after an overnight incubation. For application on embryos, defects in development and the percent that survive to adulthood are determined. For larvae, defects in behavior, locomotion, and molting may be observed. For application on adults, behavior and neurological defects are observed, and effects on fertility are noted.
For a chronic exposure assay, adults are placed on vials containing the compounds for 48 hours, then transferred to a clean container and observed for fertility, neurological defects, and death.
Assay of Compounds on Cell Cultures
Compounds that modulate (e.g. block or enhance) dmVEGFR activity may also be assayed using cell culture. For example, the effect of exogenously added compounds cells expressing dmVEGFR may be screened for their ability to modulate the activity of dmVEGFR genes based upon measurements of cell proliferation. Assays for changes in kinase activity can be performed on cultured cells expressing endogenous normal or mutant dmVEGFRs. Such studies also can be performed on cells transfected with vectors capable of expressing the dmVEGFRs, or functional domains of one of the dmVEGFRs, in normal or mutant form. In addition, to enhance the signal measured in such assays, cells may be cotransfected with genes encoding dmVEGFR proteins.
For example, binding assays with the soluble dmVEGFRs generated from cells expressing the receptors can be performed essentially as described by Piossek et al., J. Biol. Chem. (1999) 274:5612-5619. Once it has been determined that a compound is able to bind to the receptor, it can then be tested for its ability to induce endothelial cell proliferation using methods described by Cao et al. (PNAS (1998) 95:14389-14394). Briefly, endothelial cells expressing dmVEGFR are seeded in single wells of multi-well culture plate, compounds of various concentrations are added in triplicate to the wells containing the cells. Cells are then counted to check for proliferation.
Identification of small molecules and compounds as potential pharmaceutical compounds from large chemical libraries requires high-throughput screening (HTS) methods (Bolger, Drug Discovery Today (1999) 4:251-253). Several of the assays mentioned herein can lend themselves to such screening methods. For example, cells or cell lines expressing wild type or mutant dmVEGFR protein or its fragments, and a reporter gene can be subjected to compounds of interest, and depending on the reporter genes, interactions can be measured using a variety of methods such as color detection, fluorescence detection (e.g. GFP), autoradiography, scintillation analysis, etc. Compounds that selectively modulate dmVEGFR activity are identified as potential drug candidates having dmVEGFR specificity.
Both genetically modified animal models (i.e. in vivo models), such as C. elegans and Drosophila, and in vitro models such as genetically engineered cell lines expressing or mis-expressing dmVEGFR pathway genes, are useful for the functional analysis of these proteins. Model systems that display detectable phenotypes, can be used for the identification and characterization of dmVEGFR pathway genes or other genes of interest and/or phenotypes associated with the mutation or mis-expression of dmVEGFR pathway proteins. The term xe2x80x9cmis-expressionxe2x80x9d as used herein encompasses mis-expression due to gene mutations. Thus, a mis-expressed dmVEGFR pathway protein may be one having an amino acid sequence that differs from wild-type (i.e. it is a derivative of the normal protein). A mis-expressed dmVEGFR pathway protein may also be one in which one or more amino acids have been deleted, and thus is a xe2x80x9cfragmentxe2x80x9d of the normal protein. As used herein, xe2x80x9cmis-expressionxe2x80x9d also includes ectopic expression (e.g. by altering the normal spatial or temporal expression), over-expression (e.g. by multiple gene copies), underexpression, non-expression (e.g. by gene knockout or blocking expression that would otherwise normally occur), and further, expression in ectopic tissues. As used in the following discussion concerning in vivo and in vitro models, the term xe2x80x9cgene of interestxe2x80x9d refers to a dmVEGFR pathway gene, or any other gene involved in regulation or modulation, or downstream effector of the dmVEGFR pathway.
The in vivo and in vitro models may be genetically engineered or modified so that they 1) have deletions and/or insertions of one or more dmVEGFR pathway genes, 2) harbor interfering RNA sequences derived from dmVEGFR pathway genes, 3) have had one or more endogenous dmVEGFR pathway genes mutated (e.g. contain deletions, insertions, rearrangements, or point mutations in dmVEGFR gene or other genes in the pathway), and/or 4) contain transgenes for mis-expression of wild-type or mutant forms of such genes. Such genetically modified in vivo and in vitro models are useful for identification of genes and proteins that are involved in the synthesis, activation, control, etc. of dmVEGFR pathway gene and/or gene products, and also downstream effectors of dmVEGFR function, genes regulated by dmVEGFR, etc. The model systems can also be used for testing potential pharmaceutical compounds that interact with the dmVEGFR pathway, for example by administering the compound to the model system using any suitable method (e.g. direct contact, ingestion, injection, etc.) and observing any changes in phenotype, for example defective movement, lethality, etc. Various genetic engineering and expression modification methods which can be used are well-known in the art, including chemical mutagenesis, transposon mutagenesis, antisense RNAi, dsRNAi, and transgene-mediated mis-expression.
Generating Loss-of-function Mutations by Mutagenesis
Loss-of-function mutations in an invertebrate metazoan dmVEGFR gene can be generated by any of several mutagenesis methods known in the art (Ashburner, In Drosophila melanogaster: A Laboratory Manual (1989), Cold Spring Harbor, N.Y., Cold Spring Harbor Laboratory Press: pp. 299-418; Fly pushing: The Theory and Practice of Drosophila melanogaster Genetics (1997) Cold Spring Harbor Press, Plainview, N.Y.; The nematode C. elegans (1988) Wood, Ed., Cold Spring Harbor Laboratory Press, Cold Spring harbor, N.Y.). Techniques for producing mutations in a gene or genome include use of radiation (e.g., X-ray, UV, or gamma ray); chemicals (e.g., EMS, MMS, ENU, formaldehyde, etc.); and insertional mutagenesis by mobile elements including dysgenesis induced by transposon insertions, or transposon-mediated deletions, for example, male recombination, as described below. Other methods of altering expression of genes include use of transposons (e.g., P element, EP-type xe2x80x9coverexpression trapxe2x80x9d element, mariner element, piggybac transposon, hermes, minos, sleeping beauty, etc.) to misexpress genes; gene targeting by homologous recombination; antisense; double-stranded RNA interference; peptide and RNA aptamers; directed deletions; homologous recombination; dominant negative alleles; and intrabodies.
Transposon insertions lying adjacent to a gene of interest can be used to generate deletions of flanking genomic DNA, which if induced in the germline, are stably propagated in subsequent generations. The utility of this technique in generating deletions has been-demonstrated and is well-known in the art. One version of the technique using collections of P element transposon induced recessive lethal mutations (P lethals) is particularly suitable for rapid identification of novel, essential genes in Drosophila (Cooley et al., Science (1988) 239:1121-1128; Spralding et al., PNAS (1995) 92:0824-10830). Since the sequence of the P elements are known, the genomic sequence flanking each transposon insert is determined either by plasmid rescue (Hamilton et al., PNAS (1991) 88:2731-2735) or by inverse polymerase chain reaction. A more recent version of the transposon insertion technique in male Drosophila using P elements is known as P-mediated male recombination (Preston and Engels, Genetics (1996) 144:1611-1638).
Gene targeting approaches using homologous recombination have proven to be successful in Drosophila (Rong and Golic, Science (2000) 288:2013-20018) and potentially provide a general method of generating directed mutations in any gene-of-interest. This method uses broken-ended extrachromosomal DNA, created in vivo, to produce homology-directed changes in a target locus. First, a xe2x80x9ctargeting constructxe2x80x9d is designed for the gene-of-interest which allows the replacement of the normal endogenous gene with a specifically designed mutation, such as a deletion, insertion or point mutation, via homologous recombination. The targeting construct is typically carried in an appropriate transposon-mediated transgenesis vector (e.g. P element-, piggyBac-, hermes-, minos-, or mariner-based vectors) which inserts the targeting construct randomly within the genome of the organism. The targeting construct is converted to a recombinogenic extrachromosomal form by inducing the expression of separate transgenes encoding a site-specific recombinase (e.g. FLP, cre, Kw, etc.) which excises the targeting construct, and a rare-cutting site-specific endonuclease (e.g. SceI, CreI, HO, etc.) which generates recombinogenic ends that direct homologous recombination and gene replacement of the endogenous locus. Though this method has only been shown to work in Dros, it has application to worms, other animals, plants, algae etc.
Generating Loss-of-function Phenotypes Using RNA-based Methods
dmVEGFR genes may be identified and/or characterized by generating loss-of-function phenotypes in animals of interest through RNA-based methods, such as antisense RNA (Schubiger and Edgar, Methods in Cell Biology (1994) 44:697-713). One form of the antisense RNA method involves the injection of embryos with an antisense RNA that is partially homologous to the gene of interest (in this case the dmVEGFR gene). Another form of the antisense RNA method involves expression of an antisense RNA partially homologous to the gene of interest by operably joining a portion of the gene of interest in the antisense orientation to a powerful promoter that can drive the expression of large quantities of antisense RNA, either generally throughout the animal or in specific tissues. Antisense RNA-generated loss-of-function phenotypes have been reported previously for several Drosophila genes including cactus, pecanex, and Krippel (LaBonne et al., Dev. Biol. (1989) 136(1): 1-16; Schuh and Jackle, Genome (1989) 31(1):422-425; Geisler et al., Cell (1992) 71(4):613-621).
Loss-of-function phenotypes can also be generated by cosuppression methods (Bingham Cell (1997) 90(3):385-387; Smyth, Curr. Biol. (1997) 7(12):793-795; Que and Jorgens Genet. (1998) 22(1):100-109). Cosuppression is a phenomenon of reduced gene expression produced by expression or injection of a sense strand RNA corresponding to a partial segment of the gene of interest. Cosuppression effects have been employed extensively in plants and C. elegans to generate loss-of-function phenotypes, and there is a single report of cosuppression in Drosophila, where reduced expression of the Adh gene was induced from a white-Adh transgene using cosuppression methods (Pal-Bhadra et al., Cell (1997) 90(3):479-490).
Another method for generating loss-of-function phenotypes is by double-stranded RNA interference (dsRNAi). This method is based on the interfering properties of double-stranded RNA derived from the coding regions of gene, and has proven to be of great utility in genetic studies of C. elegans (Fire et al., Nature (1998) 391:806-811), and can also be used to generate loss-of-function phenotypes in Drosophila (Kennerdell and Carthew, Cell (1998) 95:1017-1026; Misquitta and Patterson PNAS (1999) 96:1451-1456). In one example of this method, complementary sense and antisense RNAs derived from a substantial portion of a gene of interest, such as dmVEGFR gene, are synthesized in vitro. The resulting sense and antisense RNAs are annealed in an injection buffer, and the double-stranded RNA injected or otherwise introduced into animals (such as in their food or by soaking in the buffer containing the RNA). Progeny of the injected animals are then inspected for phenotypes of interest (PCT publication no. WO99/32619).
Generating Loss-of-function Phenotypes Using Peptide and RNA Aptamers
Another method for generating loss-of-function phenotypes is by the use of peptide aptamers, which are peptides or small polypeptides that act as dominant inhibitors of protein function. Peptide aptamers specifically bind to target proteins, blocking their function ability (Kolonin and Finley, PNAS (1998) 95:14266-14271). Due to the highly selective nature of peptide aptamers, they may be used not only to target a specific protein, but also to target specific functions of a given protein (e.g. kinase function). Further, peptide aptamers may be expressed in a controlled fashion by use of promoters which regulate expression in a temporal, spatial or inducible manner. Peptide aptamers act dominantly; therefore, they can be used to analyze proteins for which loss-of-function mutants are not available.
Peptide aptamers that bind with high affinity and specificity to a target protein may be isolated by a variety of techniques known in the art. In one method, they are isolated from random peptide libraries by yeast two-hybrid screens (Xu et al., PNAS (1997) 94:12473-12478). They can also be isolated from phage libraries (Hoogenboom et al., Immunotechnology (1998) 4:1-20) or chemically generated peptides/libraries.
RNA aptamers are specific RNA ligands for proteins, that can specifically inhibit protein function of the gene (Good et al., Gene Therapy (1997) 4:45-54; Ellington. et al., Biotechnol. Annu. Rev. (1995) 1:185-214). In vitro selection methods can be used to identify RNA aptamers having a selected specificity (Bell et al., J. Biol. Chem. (1998) 273:14309-14314). It has been demonstrated that RNA aptamers can inhibit protein function in Drosophila (Shi et al., Proc. Natl. Acad. Sci USA (19999) 96:10033-10038). Accordingly, RNA aptamers can be used to decrease the expression of dmVEGFR protein or derivative thereof, or a protein that interacts with the dmVEGFR protein.
Transgenic animals can be generated to test peptide or RNA aptamers in vivo (Kolonin, MG, and Finley, RL, Genetics, 1998 95:4266-4271). For example, transgenic Drosophila lines expressing the desired aptamers may be generated by P element mediated transformation (discussed below). The phenotypes of the progeny expressing the aptamers can then be characterized.
Generating Loss of Function Phenotypes Using Intrabodies
Intracellularly expressed antibodies, or intrabodies, are single-chain antibody molecules designed to specifically bind and inactivate target molecules inside cells. Intrabodies have been used in cell assays and in whole organisms such as Drosophila (Chen et al., Hum. Gen. Ther. (1994) 5:595-601; Hassanzadeh et al., Febs Lett. (1998) 16(1, 2):75-80 and 81-86). expression vectors can be constructed with intrabodies that react specifically with dmVEGFR protein. These vectors can be introduced into model organisms and studied in the same manner as described above for aptamers.
Transgenesis
Typically, transgenic animals are created that contain gene fusions of the coding regions of the dmVEGFR gene (from either genomic DNA or cDNA) or genes engineered to encode antisense RNAs, cosuppression RNAs, interfering dsRNA, RNA aptamers, peptide aptamers, or intrabodies operably joined to a specific promoter and transcriptional enhancer whose regulation has been well characterized, preferably heterologous promoters/enhancers (i.e. promoters/enhancers that are non-native to the dmVEGFR pathway genes being expressed).
Methods are well known for incorporating exogenous nucleic acid sequences into the genome of animals or cultured cells to create transgenic animals or recombinant cell lines. For invertebrate animal models, the most common methods involve the use of transposable elements. There are several suitable transposable elements that can be used to incorporate nucleic acid sequences into the genome of model organisms. Transposable elements are particularly useful for inserting sequences into a gene of interest so that the encoded protein is not properly expressed, creating a xe2x80x9cknock-outxe2x80x9d animal having a loss-of-function phenotype. Techniques are well-established for the use of P element in Drosophila (Rubin and Spradling, Science (1982) 218:348-53; U.S. Pat. No. 4,670,388) and Tc1 in C. elegans (Zwaal et al., Proc. Natl. Acad. Sci. U.S.A. (1993) 90:7431-7435; and Caenorhabditis elegans: Modem Biological Analysis of an Organism (1995) Epstein and Shakes, Eds.). Other Tc1-like transposable elements can be used such as minos, mariner and sleeping beauty. Additionally, transposable elements that function in a variety of species, have been identified, such as PiggyBac (Thibault et al., Insect Mol Biol (1999) 8(1): 119-23), hobo, and hermes.
P elements, marked P elements, or piggybac elements are preferred for the isolation of loss-of-function mutations in Drosophila dmVEGFR genes because of the precise molecular mapping of these genes, depending on the availability and proximity of preexisting insertions for use as a localized transposon source (Hamilton and Zinn, Methods in Cell Biology (1994) 44:81-94; and Wolfner and Goldberg, Methods in Cell Biology (1994) 44:33-80). Typically, transposable elements are used which contain one or more elements that allow detection of animals containing the transposable element. Most often, marker genes are used that affect the eye color of Drosophila, such as derivatives of the Drosophila white or rosy genes (Rubin and Spradling, Science (1982) 218(4570):348-353; and Klemenz et al., Nucleic Acids Res. (1987) 15(10):3947-3959). However, in principle, any gene can be used as a marker that causes a reliable and easily scored phenotypic change in transgenic animals. Various other markers include bacterial plasmid sequences having selectable markers such as ampicillin resistance (Steller and Pirrotta, EMBO. J. (1985) 4:167-171); and lacZ sequences fused to a weak general promoter to detect the presence of enhancers with a developmental expression pattern of interest (Bellen et al., Genes Dev. (1989) 3(9):1288-1300).
A preferred method of transposon mutagenesis in Drosophila employs the xe2x80x9clocal hoppingxe2x80x9d method described by Tower et al. (Genetics (1993) 133:347-359). Each new P insertion line can be tested molecularly for transposition of the P element into the gene of interest (e.g. dmVEGFR) by assays based on PCR. For each reaction, one PCR primer is used that is homologous to sequences contained within the P element and a second primer is homologous to the coding region or flanking regions of the gene of interest. Products of the PCR reactions are detected by agarose gel electrophoresis. The sizes of the resulting DNA fragments reveal the site of P element insertion relative to the gene of interest. Alternatively, Southern blotting and restriction mapping using DNA probes derived from genomic DNA or cDNAs of the gene of interest can be used to detect transposition events that rearrange the genomic DNA of the gene. P transposition events that map to the gene of interest can be assessed for phenotypic effects in heterozygous or homozygous mutant Drosophila.
In another embodiment, Drosophila lines carrying P insertions in the gene of interest, can be used to generate localized deletions using known methods (Kaiser, Bioassays (1990) 12(6):297-301; Harnessing the power of Drosophila genetics, In Drosophila melanogaster: Practical Uses in Cell and Molecular Biology, Goldstein and Fyrberg, Eds., Academic Press, Inc. San Diego, Calif.). This is particularly useful if no P element transpositions are found that disrupt the gene of interest. Briefly, flies containing P elements inserted near the gene of interest are exposed to a further round of transposase to induce excision of the element. Progeny in which the transposon has excised are typically identified by loss of the eye color marker associated with the transposable element. The resulting progeny will include flies with either precise or imprecise excision of the P element, where the imprecise excision events often result in deletion of genomic DNA neighboring the site of P insertion. Such progeny are screened by molecular techniques to identify deletion events that remove genomic sequence from the gene of interest, and assessed for phenotypic effects in heterozygous and homozygous mutant Drosophila.
In C. elegans, Tc1 transposable element can be used for directed mutagenesis of a gene of interest. Typically, a Tc1 library is prepared by the methods of Zwaal et al., supra and Plasterk, supra, using a strain in which the Tc1 transposable element is highly mobile and present in a high copy number. The library is screened for Tc1 insertions in the region of interest using PCR with one set of primers specific for Tc1 sequence and one set of gene-specific primers and C. elegans strains that contain Tc1 transposon insertions within the gene of interest are isolated.
In addition to creating loss-of-function phenotypes, transposable elements can be used to incorporate the gene of interest, or mutant or derivative thereof, as an additional gene into any region of an animal""s genome resulting in mis-expression (including over-expression) of the gene. A preferred vector designed specifically for misexpression of genes in transgenic Drosophila, is derived from pGMR (Hay et al., Development (1994) 120:2121-2129), is 9Kb long, and contains: an origin of replication for E. coli; an ampicillin resistance gene; P element transposon 3xe2x80x2 and 5xe2x80x2 ends to mobilize the inserted sequences; a White marker gene; an expression unit comprising the TATA region of hsp70 enhancer and the 3xe2x80x2 untranslated region of xtubulin gene. The expression unit contains a first multiple cloning site (MCS) designed for insertion of an enhancer and a second MCS located 500 bases downstream, designed for the insertion of a gene of interest. As an alternative to transposable elements, homologous recombination or gene targeting techniques can be used to substitute a gene of interest for one or both copies of the animal""s homologous gene. The transgene can be under the regulation of either an exogenous or an endogenous promoter element, and be inserted as either a minigene or a large genomic fragment. In one application, gene function can be analyzed by ectopic expression, using, for example, Drosophila (Brand et al., Methods in Cell Biology (1994) 44:635-654) or C. elegans (Mello and Fire, Methods in Cell Biology (1995) 48:451-482).
Examples of well-characterized heterologous promoters that may be used to create the transgenic animals include heat shock promoters/enhancers, which are useful for temperature induced mis-expression. In Drosophila, these include the hsp70 and hsp83 genes, and in C. elegans, include hsp 16-2 and hsp 16-41. Tissue specific promoters/enhancers are also useful, and in Drosophila, include eyeless (Mozer and Benzer, Development (1994) 120:1049-1058), sevenless (Bowtell et al., PNAS (1991) 88(15):6853-6857), and glass-responsive promoters/enhancers (Quiring et al., Science (1994) 265:785-789) which are useful for expression in the eye; and enhancers/promoters derived from the dpp or vestigal genes which are useful for expression in the wing (Staehling-Hampton et al., Cell Growth Differ. (1994) 5(6):585-593; Kim et al., Nature (1996) 382:133-138). Finally, where it is necessary to restrict the activity of dominant active or dominant negative transgenes to regions where the pathway is normally active, it may be useful to use endogenous promoters of genes in the pathway, such as the dmVEGFR pathway genes.
In C. elegans, examples of useful tissue specific promoters/enhancers include the myo-2 gene promoter, useful for pharyngeal muscle-specific expression; the hlh-1 gene promoter, useful for body-muscle-specific expression; and the gene promoter, useful for touch-neuron-specific gene expression. In a preferred embodiment, gene fusions for directing the mis-expression of dmVEGFR pathway genes are incorporated into a transformation vector which is injected into nematodes along with a plasmid containing a dominant selectable marker, such as rol-6. Transgenic animals are identified as those exhibiting a roller phenotype, and the transgenic animals are inspected for additional phenotypes of interest created by mis-expression of the dmVEGFR pathway gene.
In Drosophila, binary control systems that employ exogenous DNA are useful when testing the mis-expression of genes in a wide variety of developmental stage-specific and tissue-specific patterns. Two examples of binary exogenous regulatory systems include the UAS/GALA system from yeast (Hay et al., PNAS (1997) 94(10):5195-5200; Ellis et al., Development (1993) 119(3):855-865), and the xe2x80x9cTet systemxe2x80x9d derived from E. coli (Bello et al., Development (1998) 125:2193-2202). The UAS/GALM system is a well-established and powerful method of mis-expression in Drosophila which employs the UASG upstream regulatory sequence for control of promoters by the yeast GAL4 transcriptional activator protein (Brand and Perrimon, Development (1993) 118(2):401-15). In this approach, transgenic Drosophila, termed xe2x80x9ctargetxe2x80x9d lines, are generated where the gene of interest to be mis-expressed is operably fused to an appropriate promoter controlled by UASG. Other transgenic Drosophila strains, termed xe2x80x9cdriverxe2x80x9d lines, are generated where the GAL4 coding region is operably fused to promoters/enhancers that direct the expression of the GALA activator protein in specific tissues, such as the eye, wing, nervous system, gut, or musculature. The gene of interest is not expressed in the target lines for lack of a transcriptional activator to drive transcription from the promoter joined to the gene of interest. However, when the UAS-target line is crossed with a GAL4 driver line, mis-expression of the gene of interest is induced in resulting progeny in a specific pattern that is characteristic for that GAL4 line. The technical simplicity of this approach makes it possible to sample the effects of directed mis-expression of the gene of interest in a wide variety of tissues by generating one transgenic target line with the gene of interest, and crossing that target line with a panel of pre-existing driver lines.
In the xe2x80x9cTetxe2x80x9d binary control system, transgenic Drosophila driver lines are generated where the coding region for a tetracycline-controlled transcriptional activator (tTA) is operably fused to promoters/enhancers that direct the expression of tTA in a tissue-specific and/or developmental stage-specific manner. The driver lines are crossed with transgenic Drosophila target lines where the coding region for the gene of interest to be mis-expressed is operably fused to a promoter that possesses a tTA-responsive regulatory element. When the resulting progeny are supplied with food supplemented with a sufficient amount of tetracycline, expression of the gene of interest is blocked. Expression of the gene of interest can be induced at will simply by removal of tetracycline from the food. Also, the level of expression of the gene of interest can be adjusted by varying the level of tetracycline in the food. Thus, the use of the Tet system as a binary control mechanism for mis-expression has the advantage of providing a means to control the amplitude and timing of mis-expression of the gene of interest, in addition to spatial control. Consequently, if a gene of interest (e.g. a dmVEGFR gene) has lethal or deleterious effects when mis-expressed at an early stage in development, such as the embryonic or larval stages, the function of the gene of interest in the adult can still be assessed by adding tetracycline to the food during early stages of development and removing tetracycline later so as to induce mis-expression only at the adult stage.
Dominant negative mutations, by which the mutation causes a protein to interfere with the normal function of a wild-type copy of the protein, and which can result in loss-of-function or reduced-function phenotypes in the presence of a normal copy of the gene, can be made using known methods (Hershkowitz, Nature (1987) 329:219-222). In the case of active monomeric proteins, overexpression of an inactive form, achieved, for example, by linking the mutant gene to a highly active promoter, can cause competition for natural. substrates or ligands sufficient to significantly reduce net activity of the normal protein. Alternatively, changes to active site residues can be made to create a virtually irreversible association with a target. The extracellular domain of VEGFR may be used as a dominant negative form to inhibit activity of VEGFR. SEQ ID NOs:7 and 8 are an isolated mutant cDNA and its protein translation, respectively, representing only the extracellular portion of dmVEGFR.
Various expression analysis techniques may be used to identify genes which are differentially expressed between a cell line or an animal expressing a wild type dmVEGFR gene compared to another cell line or animal expressing a mutant dmVEGFR gene. Such expression profiling techniques include differential display, serial analysis of gene expression (SAGE), transcript profiling coupled to a gene database query, nucleic acid array technology, subtractive hybridization, and proteome analysis (e.g. mass-spectrometry and two-dimensional protein gels). Nucleic acid array technology may be used to determine a global (i.e., genome-wide) gene expression pattern in a normal animal for comparison with an animal having a mutation in dmVEGFR gene. Gene expression profiling can also be used to identify other genes (or proteins) that may have a functional relation to dmVEGFR (e.g. may participate in a signaling pathway with the dmVEGFR gene). The genes are identified by detecting changes in their expression levels following mutation, i.e., insertion, deletion or substitution in, or over-expression, under-expression, mis-expression or knock-out, of the dmVEGFR gene.
After isolation of model animals carrying mutated or mis-expressed dmVEGFR pathway genes or inhibitory RNAs, animals are carefully examined for phenotypes of interest. For analysis of dmVEGFR pathway genes that have been mutated (i.e. deletions, insertions, and/or point mutations) animal models that are both homozygous and heterozygous for the altered dmVEGFR pathway gene are analyzed. Examples of specific phenotypes that may be investigated include lethality; sterility; feeding behavior, perturbations in neuromuscular function including alterations in motility, and alterations in sensitivity to pharmaceuticals and other compounds. Some phenotypes more specific to flies include alterations in: adult behavior such as, flight ability, walking, grooming, phototaxis, mating or egg-laying; alterations in the responses of sensory organs, changes in the morphology, size or number of adult tissues such as, eyes, wings, legs, bristles, antennae, gut, fat body, gonads, trachea, and musculature; larval tissues such as mouth parts, cuticles, internal tissues or imaginal discs; alterations in production of organs and tissues such as hemopoeisis, or larval behavior such as feeding, molting, crawling, or puparian formation; or developmental defects in any germline or embryonic tissues. Some phenotypes more specific to nematodes include: locomotory, egg laying, chemosensation, male mating, and intestinal expulsion defects. In various cases, single phenotypes or a combination of specific phenotypes in model organisms might point to specific genes or a specific pathway of genes, which facilitate the cloning process.
Genomic sequences containing a dmVEGFR pathway gene can be used to confirm whether an existing mutant insect or worm line corresponds to a mutation in one or more dmVEGFR pathway genes, by rescuing the mutant phenotype. Briefly, a genomic fragment containing the dmVEGFR pathway gene of interest and potential flanking regulatory regions can be subcloned into any appropriate insect (such a""s Drosophila) or worm (such as C. elegans) transformation vector, and injected into the animals. For Drosophila, an appropriate helper plasmid is used in the injections to supply transposase for transposon-based vectors. Resulting germline transformants are crossed for complementation testing to an existing or newly created panel of Drosophila or C. elegans lines whose mutations have been mapped to the vicinity of the gene of interest (Fly Pushing: The Theory and Practice of Drosophila Genetics, supra; and Caenorhabditis elegans: Modern Biological Analysis of an Organism (1995), Epstein and Shakes, eds.). If a mutant line is discovered to be rescued by this genomic fragment, as judged by complementation of the mutant phenotype, then the mutant line likely harbors a mutation in the dmVEGFR pathway gene. This prediction can be further confirmed by sequencing the dmVEGFR pathway gene from the mutant line to identify the lesion in the dmVEGFR pathway gene.
The characterization of new phenotypes created by mutations or misexpression in dmVEGFR genes enables one to test for genetic interactions between dmVEGFR genes and other genes that may participate in the same, related, or interacting genetic or biochemical pathway(s). Individual genes can be used as starting points in large-scale genetic modifier screens as described in more detail below. Alternatively, RNAi methods can be used to simulate loss-of-function mutations in the genes being analyzed. It is of particular interest to investigate whether there are any interactions of dmVEGFR genes with other well-characterized genes, particularly genes involved in endothelial cell signaling with kinase activity.
Genetic Modifier Screens
A genetic modifier screen using invertebrate model organisms is a particularly preferred method for identifying genes that interact with dmVEGFR genes, because large numbers of animals can be systematically screened making it more possible that interacting genes will be identified. In Drosophila, a screen of up to about 10,000 animals is considered to be a pilot-scale screen. Moderate-scale screens usually employ about 10,000 to about 50,000 flies, and large-scale screens employ greater than about 50,000 flies. In a genetic modifier screen, animals having a mutant phenotype due to a mutation in or misexpression of one or more dmVEGFR genes are further mutagenized, for example by chemical mutagenesis or transposon mutagenesis.
The procedures involved in typical Drosophila genetic modifier screens are well-known in the art (Wolfner and Goldberg, Methods in Cell Biology (1994) 44:33-80; and Karim et al., Genetics (1996) 143:315-329). The procedures used differ depending upon the precise nature of the mutant allele being modified. If the mutant allele is genetically recessive, as is commonly the situation for a loss-of-function allele, then most typically males, or in some cases females, which carry one copy of the mutant allele are exposed to an effective mutagen, such as EMS, MMS, ENU, triethylamine, diepoxyalkanes, ICR-170, formaldehyde, X-rays, gamma rays, or ultraviolet radiation. The mutagenized animals are crossed to animals of the opposite sex that also carry the mutant allele to be modified. In the case where the mutant allele being modified is genetically dominant, as is commonly the situation for ectopically expressed genes, wild type males are mutagenized and crossed to females carrying the mutant allele to be modified.
The progeny of the mutagenized and crossed flies that exhibit either enhancement or suppression of the original phenotype are presumed to have mutations in other genes, called xe2x80x9cmodifier genesxe2x80x9d, that participate in the same phenotype-generating pathway. These progeny are immediately crossed to adults containing balancer chromosomes and used as founders of a stable genetic line. In addition, progeny of the founder adult are retested under the original screening conditions to ensure stability and reproducibility of the phenotype. Additional secondary screens may be employed, as appropriate, to confirm the suitability of each new modifier mutant line for further analysis.
Standard techniques used for the mapping of modifiers that come from a genetic screen in Drosophila include meiotic mapping with visible or molecular genetic markers; male-specific recombination mapping relative to P-element insertions; complementation analysis with deficiencies, duplications, and lethal P-element insertions; and cytological analysis of chromosomal aberrations (Fly Pushing: Theory and Practice of Drosophila Genetics, supra; Drosophila: A Laboratory Handbook, supra). Genes corresponding to modifier mutations that fail to complement a lethal P-element may be cloned by plasmid rescue of the genomic sequence surrounding that P-element. Alternatively, modifier genes may be mapped by phenotype rescue and positional cloning (Sambrook et al., supra).
Newly identified modifier mutations can be tested directly for interaction with other genes of interest known to be involved or implicated with dmVEGFR genes using methods described above. Also, the new modifier mutations can be tested for interactions with genes in other pathways that are not believed to be related to receptor tyrosine kinase signaling (e.g. nanos in Drosophila). New modifier mutations that exhibit specific genetic interactions with other genes implicated in neuronal signaling, but not interactions with genes in unrelated pathways, are of particular interest.
The modifier mutations may also be used to identify xe2x80x9ccomplementation groupsxe2x80x9d. Two modifier mutations are considered to fall within the same complementation group if animals carrying both mutations in trans exhibit essentially the same phenotype as animals that are homozygous for each mutation individually and, generally are lethal when in trans to each other (Fly Pushing: The Theory and Practice of Drosophila Genetics, supra). Generally, individual complementation groups defined in this way correspond to individual genes.
When dmVEGFR modifier genes are identified, homologous genes in other species can be isolated using procedures based on cross-hybridization with modifier gene DNA probes, PCR-based strategies with primer sequences derived from the modifier genes, and/or computer searches of sequence databases. For therapeutic applications related to the function of dmVEGFR genes, human and rodent homologues of the modifier genes are of particular interest.
Although the above-described Drosophila genetic modifier screens are quite powerful and sensitive, some genes that interact with dmVEGFR genes may be missed in this approach, particularly if there is functional redundancy of those genes. This is because the vast majority of the mutations generated in the standard mutagenesis methods will be loss-of-function mutations, whereas gain-of-function mutations that could reveal genes with functional redundancy will be relatively rare. Another method of genetic screening in Drosophila has been developed that focuses specifically on systematic gain-of-function genetic screens (Rorth et al., Development (1998) 125:1049-1057). This method is based on a modular mis-expression system utilizing components of the GAL4JUAS system (described above) where a modified P element, termed an xe2x80x9cenhanced Pxe2x80x9d (EP) element, is genetically engineered to contain a GAM4-responsive UAS element and promoter. Any other transposons can also be used for this system. The resulting transposon is used to randomly tag genes by insertional mutagenesis (similar to the method of P element mutagenesis described above). Thousands of transgenic Drosophila strains, termed EP lines, can be generated, each containing a specific UAS-tagged gene. This approach takes advantage of the preference of P elements to insert at the 5xe2x80x2-ends of genes. Consequently, many of the genes that are tagged by insertion of EP elements become operably fused to a GAL4-regulated promoter, and increased expression or mis-expression of the randomly tagged gene can be induced by crossing in a GAL4 driver gene.
Systematic gain-of-function genetic screens for modifiers of phenotypes induced by mutation or mis-expression of a dmVEGFR gene can be performed by crossing several thousand Drosophila EP lines individually into a genetic background containing a mutant or mis-expressed dmVEGFR gene, and further containing an appropriate GALA driver transgene. It is also possible to remobilize the EP elements to obtain novel insertions. The progeny of these crosses are then analyzed for enhancement or suppression of the original mutant phenotype as described above. Those identified as having mutations that interact with the dmVEGFR gene can be tested further to verify the reproducibility and specificity of this genetic interaction. EP insertions that demonstrate a specific genetic interaction with a mutant or mis-expressed dmVEGFR gene, have a physically tagged dmVEGFR which can be identified and sequenced using PCR or hybridization screening methods, allowing the isolation of the genomic DNA adjacent to the position of the EP element insertion.